Prostate-specific membrane antigen (PSMA) is a well-characterized glycoprotein that is expressed in normal individuals as a cytoplasmic protein in the prostate epithelial cells. In prostate carcinomas, however, its gene undergoes alternative splicing, and as a result of this, exists as a Type II membrane glycoprotein with a large and very antigenic extracellular region. Recent data indicates that PSMA is also abundantly expressed on the neovasculature of many other important tumors. This unique pattern of expression makes PSMA a compelling target for active immunotherapy or vaccine development not only for prostate cancer, but also for other tumors. Effective vaccination against self-antigens, such as PSMA, will need to overcome the existing peripheral tolerance to the protein. In our experience, the most effective vaccines for breaking tolerance in man comprise a tumor-associated antigen conjugated to a large and highly immunogenic carrier protein, such as keyhole limpet hemocyanin (KLH) in combination with an effective immunostimulatory adjuvant. This strategy has proven effective in inducing tumor-specific immunity and objective clinical responses in man. The overall goal of this proposal is to develop the best possible protein vaccine for prostate cancer immunotherapy. The studies employ a novel recombinant soluble PSMA protein (rsPSMA) that reproduces the native tertiary and quartenary structure of tumor-associated PSMA. This vaccine will be used alone and/or as the potential protein boost component in heterologous prime-boost regimens. During Phase I of the project we critically evaluate the potency of rsPSMA-KLH conjugate vaccines v. traditional adjuvanted protein vaccines prior to initiating human clinical trials with the most promising approach. In this Phase I project, we develop highly optimized rsPSMA-conjugates that maximize incorporation of native, biologically active rsPSMA. For our in vivo studies we will use a human HLA-A2 transgenic mouse model, which we believe is the currently best available in vivo model for the proposed comparative vaccine studies. These activities are intended to identify a PSMA-KLH conjugate vaccine that is clearly superior to adjuvanted PSMA alone. These studies will also accelerate our development of assays for monitoring the cellular and humoral responses elicited in subsequent clinical studies. Overall, we will utilize state-of-the-art technologies to optimally exploit PSMA as a promising new target for active immunotherapy of prostate cancer.